Recent developments in production and purification of malaria antigens: In vitro propagation of Plasmodium falciparum for merozoite antigens*

Bulletmn of ihe World Health Orga8nzation, 57 (Suppl 1): 33-36 (1979) In vitro propagation of Plasmodium falciparum for merozoite antigens* R. G. BRACKETT,I G. C. COLE,2 T. J. GREEN,3 & R. L. JACOBS' A system with continuousflow ofmedium that uses disposable tissue cultureflasksfor the in vitro cultivation of P. falciparum is described. This scaled-up modification of the Trager and Jensen method gives merozoiteyields sufficientfor experimental vaccine studies in animals. A method of increasing the yield of merozoites by decreasing the pH of the medium is presented. The malaria immunology research efforts in this laboratory have been directed towards the develop- ment of a merozoite vaccine. We have been growing Plasmodium faciparum in Petri dishes in candle jars and in flow-flasks in a manner essentially the same as that first described by Trager & Jensen (1). We modified and scaled up their flow system so as to be able to produce yields of merozoites sufficient to pre- pare experimental vaccines for animal inoculation. This culture system and a method to increase the yield of merozoites will be descnbed. MATERIALS AND METHODS Flow-flask apparatus Fig. 1 presents a schematic diagram of the culture system. Commercially available, disposable, sterile polystyrene tissue culture flasksa are used as culture vessels. Four holes are made in the top of the flask at appropriate sites with a hot cork-borer. One-hole, silicone rubber stoppers bearing 3-mm or 5-mm (j- inch or 4-inch) OD stainless steel cannulas serve as ports through which media and gas can be delivered and removed via silicone rubber tubing. A refriger- ated bath maintains the medium at 4 IC. It is pumped * From Biological Research and Development, Warner-Lambert! Parke-Davis, Joseph Campau at the River, Detroit, MI 48232, USA This research has been supported in part by contracts AID/ta-C-1258 and AID/ta-C-1440 with the United States Agency for Internalional Dcvelopmnenl. Director, Cliucal Immunology. Research Associate. ' SenLorScientist. Scientist. a From Corning Glass Works, Corning. Ncw York. NY. USA b From Manostat Corp., Ncw York, NY, USA From Harvard Apparatus Co lMillis, MA, USA. continuously by means of a Manostatb or a Harvard pumpI into a port at the back of the flask and out of a port located over the upward sloped neck of the flask. The afferent cannula is positioned so as to deliver fresh medium above the surface of the culture fluid. The level of the medium is adjusted by the position of the efferent cannula. Effluent medium is pumped from the flow vessel into a graduated Erlenmeyer flask. Gaseous atmnosphere A compressed mixture of oxygen, carbon dioxide, and nitrogen is humidified, sterile-filtered and passed in through a port at the back of the flask and out from a port at the front. Efferent gas is led to a gas trap and then into a cylinder containing disinfectant through which the gas is bubbled. This arrangement prevents a back-flow of non-sterile air into the flask, and causes a slight back-pressure, ensuring an even distribution of gas. The gas flow can be readily observed and the potential for contamination of the 370C incubator and room is reduced. When several flasks are used, the gas mixture is passed through the flasks by con- necting the efferent line of the first flask to the afferent of the next and so on. Erythrocytes and medium Human red blood cells, typeAB or 0, are aged for at least 1 week before being used. The cells are mixed with Pfa(ciparum-infected cells from Petri-dish or flow-flask cultures and suspended at concentrations of 12-50% in RPM1 1640 medium with 100 ml of human type AB or 0 serum per litre. The erythrocytes are allowed to settle and form a shallow layer on the bottom of the flask before pumping of the medium is begun. Eruthrocytes are removed from the flask 2-3 3866 -33- R. G. BRXCKETT ET AL. F ILTERF ILTER 370°C INCUBATOR FLOW FLASK APPARATUS Fig. 1. Diagrammatic representation of the flow-flask apparatus for in vitro propagation of P. falciparum. times per week, centrifuged, mixed with uninfected red blood cells and medium, and returned to the flask. Parasites The African strain ofP.falciparum, FCR-3, which is currently receiving most emphasis, was received from Dr Trager and Dr Jensen in October 1977. However, much of the culture system development work was done witb the FVO South-East Asian strain which we obtained 9 months earlier. RESULTS The flow-flask system has been established in our laboratory by Dr George Cole and Dr Richard Jacobs, who examined the effect of numerous culture vari- ables on parasite growth. Surface area, haematocrit, andflow rate Early experiments were performed in 25-cm2 flasks with a volume ofmedium plus cells of 3 ml and a flow rate of the medium of 2 nil/h. RBC concentrations of 50, 25, and 12.5% were examined to determine the influence of baematocrit on percentage parasitaemia. In general, when other parameters were held con- stant, parasitaemia was inversely proportional to red blood cell concentration. The total yield of parasites per fask did not vary much over the range of erythro- cyte concentrations tested. When a flask with a surface area of 75 cm2 was used and the volume of cells plus medium was in- creased to 9 ml, the 2 ml/h flow rate became limiting. If the flow rate was increased to 4.5 ml/h, parasite growth was equivalent to that in the 25-cm2 flask. Thus, we have achieved parasitaemia of 3-25% depending on the starting parasitaemia, haematocrit, and flow rate of medium. Expressed in another way, up to 6-fold increases in the number of parasitized erythrocytes have been achieved during 2- and 3-day growth periods. Gaseous atmosphere Carbon dioxide concentrations from 3 to 7To and oxygen concentrations from 5 to 10%o did not signifi- cantly affect the percentage parasitacmia in this cul- ture system, irrespective of whether Tygon or silicone rubber tubing was used for gas delivery. A mixture of 3% C02: 10% 02 87% N2 is now used in our cul- tures. Critical factors in the selection of optimal culture conditionis are the solubility of the gases in the 34 IN IYTRO PROPAGATION OF P. F4LCJP4RUM 35 medium, depth of medium, concentration of erythro- cytes (and hence the depth of the settled erythrocyte layer) and the dissolved gas gradient that develops. It is possible to increase the availability of the gas mixture to the infected erythrocytes by increasing the surface area of the liquid-gas interface. To this end, we have examined the suitability of Teflon FEP fluo- rocarbon film bags as static culture vessels for P.falci- parum. Bags made from thin layers of this material are highly permeable to carbon dioxide, oxygen, and nitrogen while being relatively impermeable to water vapour and have been used for mammalian tissue cultures (2). When FEP bag cultures were incubated for 3 days at 17, 12, 7, or 2o 0,2 no differences in the rates of parasite multiplication were observed; but 21 02 did not appear to maintain erythrocytes as well as the higher 02 concentrations. pH An unexpected observation was made on slides pre- pared from red blood cells in FEP bag cultures in which the medium had not been renewed. Large num- bers of merozoites were seen to accumulate in one or two days. Dr Ted Green followed up this finding and found that, while adequate levels of glucose remained in the medium, lactic acid accumulation had caused the pH to fall. To study the influence of pH on mero- zoite accumulation, replicate Petri-dish cultures were prepared with medium adjusted to initial pH levels from 6.8 to 7.2. A starting parasitaernia of 1%a was selected and the cultures placed in candle jars at 37 °C. Table I shows the mean distribution of mero- zoites and ring forms per 100 parasite-associated red cells during this 3-day experiment. The values shown in the table are mean counts from 9 or 6 plates per pH level and show highly significant changes in the ratio Table 1. Mean numbers of merozoites and ring forms per 100 parasite-associated red cells in media of differernt pH (Petri dish culture in candle jars) pH of medium 68 69 71) 71 7.2 Day 0 merozohes 12 06 0 6 13 13 nng torns 19 4 26 4 20 6 24 5 210(n X 9) DaY I merozotes 42 3 35 7 196 7 2 3 9 nng forrn 4 3 56 160 29.8 253 In = 9) Day 2 meozones 582 48.0 34 5 12.0 4.7 uing forms 3 5 2.3 153 298 363 tn - 61 of the stages foiund in the cultures each day. The potential for utilizing this simple manipulation to increase the yield of merozoites from an asyn- chronous P.falciparum culture was determined in the flow-flask system described earlier. It was found that the total yield ofmerozoites from a 75-c_M2 flow-flask was 2-3 times greater when the pH was reduced to 6.8 for 24 h as compared with controls maintained at pH 7.2 (for example 1.6 x 10J compared with 4.76 x 10'). The merozoites maintain a healthy appearance and are most frequently found associated with the surface of red cells or free in the medium. The anti- genicity of merozoites obtained in this fashion is being tested. By altering the pH of an asynchronous culture or one in which synchrony has been induced by adding sorbitol, for example, it is possible to increase sub- stantially the yield of rnerozoites by interruption of the reinfection process. Such a manipulation of pH is simple to accomplish in flow-flasks and in the Teflon bags of the IL-410 Cell Culture System" which is currently under evaluation in our laboratory. Characteristics ofthe cultured parasites We have maintained the FCR-3 strain in continuous culture for 285 days and this, together with the previous period of growth at Rockefeller University, amounts to more than 2j years of in vitro cultivation. What effect this period in culture has exerted on the antigenic characteristics of the strain remains to be determined. The culture system is devoid of anti- bodies that mighi. mi vivo, exert selection pressures for particular variants (3). Clearly, infectivity is maintained in this culture system. The FCR-3 strain propagated in human eryth- rocytes for 14 months caused chronic. self-limiting malaria in Aorus trivirgarus grrseitneinbra during the fiist three passages, but in subsequent passages an acute fulminating infection was produced which ended with death of the monkeys. Thus, it seems prudent to suggest that when P.fal- ciparini cultures are used for the produiction of ex- perimental human vaccines, early passage levels should be chosen to minimize the possibility of cul- ture-induced variation. A large supply of parasitized erythrocytes snould be frozen at early passage and held in liqutid nitrogen to serve as a stock source for inutiating cultures destined for vaccine preparation to ensure uniformity of the P.falciparunm inocultim. We have experienced excellent recovery of in vitro cul- tured P.falciparum-infected erythrocytes frozen by the Rowe method (4): therefore, stock cultures are technically feasible and highly desirable. d From lnstrumeniauon Laboratories, Inc, Lexington, -MA, USA 36 R G. BRACKETT ET AL. RESUMPE CULTURE IN VITRO DE PLASMODIUMFALCIPARUM EN VUE DE LA PRODUCTION DE VACCINS EXPLRIMENTAUX A BASE D'ANTIGVNES DE MtROZOQTES En partant de la m6thode de base mise au point par Trager et Jensen, les auteurs d6crivent un systime pour la culture in vitro de Plasmodiumfalciparum qui utilise des flacons a jeter permettant un renouvellement continu du milieu. L'identifLcation des facteurs determinants pour creer des conditions de culture optimales-solubilt des gaz employes dans le milieu et taux de concentration de ceux-ci a travers le milieu selon 'Epaisseur dela couched'6rythrocytes infectfs- a amen6 A exp6rimenter, pour accroitre J'interphase de contact entre le minange gazeux et la couche d'&ythrocytes, de simples poches statiques form6es d'une pellicule plastique A forte permeabilit6 aux gaz. Dans cette derni6re exp6rience, une baisse de pH du milieu s'est accompagn6e d'une forte accumulation de merozoites en 1-2 jours. L'influence d'une variation de pH entre 6,8 et 7,2 a W 6tudi6e dans des cultures en boites de P&tri plac6es dans un dessicateur A bougie, et l'Importance relative des stades du parasite (mrnozoites/ formes annulaires) a pr&sent. de fortes variations avec les divers pH. Lors d'essaLs de modification du pH dans Le systneme dc culture en flacons A flux continu, la r6ductLon du pH A 6,8 pendant 24 heures a permis de doubler ou tnpler la production de m6rozoites. On obtient avec ces m&thodes des merozoYtes en quantites suffLsantes pour la preparation de vaccins cxpErimenraux et leur pouvoir infectant a 'et contrWl6. REFERENCES 1. TRAGER, W. & JENSEN, J. B. Science, 193: 673-675 (1976). 2. JENSEN, M. D. ET AL. Journal oftheoretical biology, 56: 443458 (1976). 3. ZUCKERMAN, A. Experimentalparasitology, 42: 374 446 (1977). 4. ROWE, A. W. ET AL. Cryobiology, 5: 119-128 (1968).